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- Perols, Anna, et al.
(författare)
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Site-specific antibody labeling by covalent photoconjugation of Z domains functionalized for alkyne-azide cycloaddition reactions
- 2015
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Ingår i: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 16:17, s. 2522-2529
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are extensively used both as research tools and in the clinics for diagnostics and therapy. For many applications the antibodies are labeled, e.g. with detection probes or cytotoxic agents that improve the therapeutic effect. Labeling is typically performed using amine-reactive probes targeting lysine residues accessible on the surface of the protein, resulting in a heterogeneously labeled antibody. One alternative strategy for site-specific labeling is to use the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc-region of IgG. By introducing the photoactivable amino acid benzoylphenylalanine (BPA) in the Z domain, a covalent bond can be formed between the Z domain and the antibody by UV irradiation, to produce a site-specifically labeled product. The Z domain with BPA in position 32, Z32BPA, was synthesized by solid phase peptide synthesis and was further functionalized for alkyne-azide cycloaddition reactions to give alkyne-Z32BPA and azide-Z32BPA for Cu(I)-catalyzed click reaction, and DBCO-Z32BPA for Cu-free strain-promoted click reaction. The reactivity of the functionalized Z domains in Cu(I)-catalyzed and strain-promoted alkyne-azide cycloaddition reactions was analyzed using MALDI-TOF MS and RP-HPLC, showing fast reaction. To further evaluate the concept, the Z32BPA variants were conjugated to the human IgG1 antibody trastuzumab and biotinylated by Cu(I)-catalyzed or strain-promoted alkyne-azide cycloaddition reactions. Western blot analysis of the biotinylated antibodies, using streptavidin-HRP for detection, demonstrated that all Z-antibody conjugates could be site-specifically labeled by the alkyne-azide cycloaddition reactions.
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